Members of the AraC protein family regulate a broad range of activities important in the pathogenicity of E. coli: bacterial stress response, invasion of intestinal mucosa and toxin release. The proteins MarA, SoxS and Rob (the latter two approximately 45% identical to MarA), are members of an AraC sub-family of transcriptional activators that mediate resistance to superoxides, organic solvents and a broad range of antibiotics in a number of gram-negative bacteria. The binding site or 'marbox' to which MarA, SoxS and Rob bind is located in the region immediately upstream of the binding signals (-10 and -35 sites) for RNA polymerase (RNP). The marbox is a highly degenerate asymmetric sequence oriented in those promoters in which it overlaps the -35 site (Class II promoters), so that the COOH-terminus of MarA lies adjacent to RNP and in the opposite orientation in those promoters where the marbox lies 15 (or 26) bp upstream of the -35 site (Class I promoters). To determine which amino acids of MarA interact with RNP, each of the amino acids has been substituted (one at a time) with alanine and the mutant MarA proteins assayed for their ability to stimulate either Class I or Class II promoters. Potential sites have been identified for Class II but not Class I promoters (Gillette, Martin & Rosner, J.M.B. [2000] 299,1245). To determine which components of RNP are important for activation by MarA, a library of sigma mutants has been screened for their ability to be stimulated by MarA and none of the mutants has been found to influence MarA activation, suggesting that the sigma subunit of RNP is not a point of contact. This latter conclusion has been further corroborated by the demonstration both in vivo and in vitro that the alternate sigma factor encoded by rpoS (sigma 38) is effective in MarA activation. A potential fourth member of the MarA, SoxS, Rob subfamily was identified on genomic sequencing of E. coli. This gene, ykgA, has been demonstrated not to activate promoters of the regulon (Martin, Gillette and Rosner, Mol. Microbiol. [2000] 37, 978. In collaboration with B. Dangi, J. L. Louis and A. M. Gronenborn, we are studying the interaction of MarA with a number of different marbox sequences. Preliminary results suggest that different interactions occur between MarA and the different binding sites.